The DPP, nationally, recruited 3,234 volunteers who received standard lifestyle recommendations and were randomly assigned to one of three interventions: intensive lifestyle with the aim of losing and maintaining 7% weight loss and achieving > 150 minutes per week of moderate intensity physical activity, metformin therapy with 850 mg twice per day, or placebo. The development of diabetes in the lifestyle intervention and metformin-treated groups were reduced by 58% and 31%, respectively, compared with the placebo group Many important issues remained unanswered, however. Specifically, whether the decrease in the development of diabetes can be sustained is unknown. Moreover, determining whether the delay or prevention of diabetes will translate into a decrease in retinopathy, nephropathy, neuropathy, and cardiovascular disease, all of which require more years to develop than the DPP period of study, is critical to establish the true impact of the DPP on public health. This long-term follow-up study of the DPP the DPPOS will address these issues. All DPP participants, whether or not they developed diabetes during the DPP, were invited to join DPPOS. 91% of the participants in the American Indian centers chose to continue in DPPOS. This was the highest percentage of any racial/ethnic group in the DPPOS. The DPPOS has continued to progress very smoothly at the American Indian sites operated by the Diabetes Epidemiology and Clinical Research Section. Retention of American Indian participants is excellent. 94% of our participants are still actively followed (defined as not having missed the last two visits), compared with 91% in the study nationally. This allows Southwestern American Indians, a population highly affected by diabetes, to participant in this major clinical trial determining long-term health benefits of diabetes prevention interventions. In the DPPOS, low vitamin B12 concentrations (<203 pg/mL) occurred more often in the metformin group than in the placebo group at 5 years (4.3 vs. 2.3%;P=.02) but not at13 years (7.4 vs.5.4%;P=12).Combined low and borderline low B12 (<298pg/mL) was more common in metformin at 5 years (19.1 vs. 9.5%;P=.01) and 13 years (20.3 vs. 15.6%;P=.02). Years of metformin use were associated with increased risk of B12 deficiency (odds ratio, B12 deficiency/year metformin use, 1.13; 95% confidence interval, 1.061.20). Anemia prevalence was higher in metformin, but did not differ by B12 status. Neuropathy prevalence was higher in metformin with low B12 levels. Long-term use of metformin in DPPOS was associated with biochemical B12 deficiency and anemia. Routine testing of vitamin B12 levels in metformin-treated patients should be considered. We performed plasma metabolite profiling in the Diabetes Prevention Program (DPP), a completed trial that randomized high-risk individuals to lifestyle, metformin, or placebo interventions. Our findings indicate betaine is a marker of diabetes risk among high-risk individuals both at baseline and during preventive interventions, and complement animal models demonstrating a direct role for betaine in modulating metabolic health.